modulation of fibroblast proliferation and inflammatory response

Received on August 22, 2005.Approved by the Consultive Council and accepted for publication on March 21, 2006. * Study conducted at the Immunopathology and Experimental Pathology Laboratory of the Centro de Biologia da Reprodução da Universidade Federal de Juiz de Fora - UFJF -Juiz de Fora (MG), Brazil.Conflict of interest: None1 Centro de Biologia da Reprodução – CBR of the Universidade Federal de Juiz de Fora. Immunopathology and Experimental Pathology Laboratory; Master’s degree student in

Brazilian Health - Universidade Federal de Juiz de Fora - UFJF - Juiz de Fora (MG), Brazil. 2 Dental surgeon of the Faculdade de Odontologia - Universidade Federal de Juiz de Fora - UFJF - Juiz de Fora (MG), Master’s degree in Laser in Dentistry from the Universidade de

São Paulo - USP - São Paulo (SP), Brazil.3 Researcher of the Centro de Biologia da Reprodução – CBR - Universidade Federal de Juiz de Fora – UFJF - Juiz de Fora (MG). Immunopathology and Experimental

Pathology Laboratory; Ph.D. in Pathology from the Universidade Federal Fluminense - UFF - Niterói (RJ), Brazil.4 Fundação Instituto Mineiro de Estudos e Pesquisas em Nefrologia – Imepen - Juiz de Fora (MG), Ph.D. in Nephrology from the Universidade Federal de São Paulo - UNIFESP

- São Paulo (SP), Brazil.5 Researcher of the Centro de Biologia da Reprodução – CBR of the Universidade Federal de Juiz de Fora - UFJF - Juiz de Fora (MG), Immunopathology and Experimental

Pathology Laboratory; Ph.D. in Pathology from the Universidade Federal Fluminense - UFF - Niterói (RJ), Brazil.

©2006 by Anais Brasileiros de Dermatologia

Modulation of fibroblast proliferation and inflammatory responseby low-intensity laser therapy in tissue repair process*

Modulação da proliferação fibroblástica e da respostainflamatória pela terapia a laser de baixa intensidade noprocesso de reparo tecidual*

Adeir Moreira Rocha Júnior1 Rodrigo Guerra de Oliveira2 Rogério Estevam Farias3

Luiz Carlos Ferreira de Andrade4 Fernando Monteiro Aarestrup5

150

An Bras Dermatol. 2006;81(2):150-6.

Clinical, Epidemiological, Laboratory and Therapeutic Investigation

Abstract: BACKGROUND - Several studies have been performed to understand the tissue repair process aswell as the possible effects of laser therapy in wound healing. OBJECTIVES - To study the behavior of skin wounds induced in the dorsal region of Wistar rats (Rattusnorvegicus), which were submitted to the low-intensity laser therapy at 3.8 J/cm2 dosage, 15mWpotency, during 15 seconds. MATERIAL AND METHODS - The animals (n=12) were divided into two groups - control and laser-treat-ed. The latter comprised three applications (immediately after surgery, 48 hours and 7 days afterinduction of surgical wounds). Ten days after surgery samples of the wounds were collected andsubmitted to histopathological and histomorphometric studies.RESULTS - Neovascularization, fibroblast proliferation and reduced inflammatory infiltrate in surgicalwound submitted to laser therapy were demonstrated. CONCLUSION - Taken together, the results suggest that low-intensity laser therapy is an effectivemethod to modulate tissue repair, thus significantly contributing to a faster and more organizedhealing process. Keywords: Laser therapy, low-level; Rats; Wound healing

Resumo: FUNDAMENTOS - Ao longo dos anos, diversos estudos têm sido realizados para compreen-der o processo de reparo tecidual, bem como os possíveis efeitos da terapia a laser no processo decicatrização de feridas. OBJETIVOS - Investigar o comportamento de feridas cutâneas provocadas na região dorsal de ratosWistar (Rattus norvegicus), que foram submetidos ao tratamento com laser de baixa intensidade,com 3,8 J/cm2 de dosagem, 15mW de potência e tempo de aplicação de 15s. MÉTODOS – Os animais (n = 12) foram divididos em dois grupos, um controle e outro tratado comlaser. Foram realizadas, no grupo tratado, três aplicações (imediatamente após o ato cirúrgico,48 horas e sete dias após a realização das feridas cirúrgicas). Dez dias após o ato cirúrgico foramcolhidas amostras das lesões de ambos os grupos para realização de estudo histopatológico e his-tomorfométrico. RESULTADOS - Foram evidenciados aumentos da neovascularização e da proliferação fibroblástica, ediminuição da quantidade de infiltrado inflamatório nas lesões cirúrgicas submetidas à terapia comlaser. CONCLUSÃO - Os resultados em conjunto sugerem que a terapia a laser de baixa intensidade é ummétodo eficaz no processo de modulação da reparação tecidual, contribuindo significativamentepara a cicatrização tecidual mais rápida e organizada. Palavras-chave: Cicatrização de feridas; Ratos; Terapia a laser de baixa intensidade


INTRODUCTIONTissue repair is a dynamic state that encompas-

ses different processes, among which inflammation,cell proliferation and synthesis of elements whichconstitute the extracellular matrix, such as collagen,elastin and reticular fibers.1 Collagen synthesis is a fastand harmonic process that starts with the interstitiallesion and extends up to the end of the healing phase,when the remodeling of the tissues occurs.2

The scarring and tissue repair processes occurafter trauma or disease.3 Wound repair and restructu-ring constitute a complex mechanism, in which seve-ral factors contribute to a variety of scarring types,such as hypertrophy, atrophy or normotrophy of theinjured area. These processes comprise three phases:inflammation, granulation and formation of the extra-cellular matrix.4 Normally, in the wound healing pro-cess, after the granulation phase starts, there is a slightpredominance of macrophages and an increase in thenumber of fibroblasts, with synthesis of a new extra-cellular matrix, and remodeling of these tissues occurswith a contraction of the granulation tissue. Duringthe matrix formation phase, the fibroblasts producegreat amounts of extracellular matrix. Collagensynthesis occurs on the 21st day after the lesion andthe return of the skin to its normal aspect on the 26th

day.5 Once the wound is resolved and involved by gra-nulation tissue, a significant decrease in macrophagesand fibroblasts occurs, and the maturation of the scarbecomes relatively acellular.6

Radiation and is based on a theory developedby physicist Albert Einstein who, in his article “ZurQuantumtheorie der Strahlung”, of 1917, presentedthe physical principles of stimulated emission (thelaser phenomenon), which is classified as “high-power” (with destructive potential) or “low-power”(without destructive potential).7 This therapy was firstused by Mester et al., who used 488 and 515nm argonlaser. Subsequently, the helium-neon (He-Ne) laserwas introduced, which emits red light with a wave-length of 632.8nm, and was replaced by a less expen-sive and more powerful device, the diode laser, with awavelength of 660-950nm.8 Experimental treatmentsin patients started in the 1970’s, following reports ofpositive results obtained by irradiation with low-inten-sity laser therapy (LILT) of cell cultures and in animalexperiments. The studies performed were insufficientto confirm the beneficial effects of LILT.9,10 effectsappeared,11 but failed due to the great number ofinterventions and to unsatisfactory quality of metho-dology.

Several studies have been conducted in orderto understand the wound healing process, aiming toclarify the different aspects of the granulation tissue,of tissue epithelization and neoformation, as well as

the possible effects of LILT in the tissue repair process.This study had the objective of evaluating, by means ofa histopathologic and histomorphometric analysis, theclinical-biological behavior of skin wounds induced inthe dorsal area of Wistar rats (Rattus norvegicus) sub-mitted to LILT. In particular, the effects of LILT onangiogenesis, fibroblast proliferation and the inflam-matory infiltrate were analyzed.

MATERIAL AND METHODSAnimals

Twelve male Wistar rats (Rattus norvegicus), sixto eight weeks old, weighing 160 to 220 g, providedby the Animal House of the Centro de Biologia daReprodução – CBR [Reproduction Biology Center] ofthe Universidade Federal de Juiz de Fora (UFJF/MG),were used. The rat housing at CBR-UFJF has largedumping containers with wire screens and twoexhausters, besides room heaters. The temperature iskept at around 22ºC, by natural ventilation in summerand with the help of heaters in winter. The lighting ismixed – natural light and fluorescent light bulbs, auto-matically controlled to get on at 6:00 a.m. and off at6:00 p.m. The animals were kept in individual poly-propylene cages, equipped with beds of selectedwood shavings, baby bottles with water and troughsfor palletized chow, under maintenance conditionswhich are in agreement with the criteria of theColégio Brasileiro de Experimentação Animal[Brazilian College of Animal Experimentation], andsubmitted to daily macroscopic evaluations in orderto observe signs indicating secondary infection.

The animals were randomly divided into twogroups, one (I) submitted to surgical skin wounds(n=6), and the other (II) to surgical skin wounds andlow-intensity laser therapy (n=6).

In the treatment protocol used for this study,group I was kept as control and, in group II, lasertreatment was performed; the whole experiment las-ted 10 days.

Experimental modelcThe dorsal area of the animals was shaved after

anesthesia with intraperitoneal ketamine (100ml/kg)+ xylazine (10mg/kg) and, by means of a punch ofapproximately 10mm in diameter, a circular fragmentof skin tissue was removed.

The treatment was carried out using low-inten-sity laser (Twin Laser) with the following characteris-tics: infrared emission laser, pulsatile, arsenium andgallium semiconductors, wavelength of 870nm, peakpower of 70mW, mean exit power of 0.5 to 3.5mW andapplication through fiber optics. The application wasdone by the scanning method in the central area of

Modulation of fibroblast proliferation and inflammatory response... 151


the wound, thus allowing its uniform treatment. Inthe control group, no kind of treatment was used. Ingroup II, the experimental wounds were submitted toLILT with the following parameters: 15mW of power,a 3.8J/cm2 dose for 15s on each one of three applica-tions, the first one immediately after surgery, the sec-ond one 48 hours after the surgical procedure, andthe third one seven days after performing the surgicallesion.

After 10 days from the surgery and after laserapplication, the animals were sacrificed with an over-dose of anesthesia with intraperitoneal ketamine(100ml/kg) + xylazine (10mg/kg). The samples fromthe skin lesions were collected so that part of the skinadjacent to the wound rims and the entire scar tissuein all its depth were included.

Histopathology and histomorphometryAll skin lesion samples obtained were fixed in

10% buffered formalin (pH 7) for at least 24 hours.After fixation, the samples were gradually dehydratedin increasing concentrations of ethanol (70% to100%), cleared in xylene, soaked and embedded inparaffin, according to routine histological methods.The paraffin-embedded fragments were cut with an“820” Spence microtome and 6?m thick sections wereobtained. The histological slides were kept in an incu-bator to dry, and then the sections were stained withhematoxylin and eosin for histological analysis.

Histomorphometry was performed usingimages captured and evaluated by a computerizedAxion Vision (Zeiss, Berlin, Germany) image capturesystem. Images were captured from four randomlychosen microscopic fields for each histological slide,using the digital camera (total enlargement 400x) ofan Axiostar Plus microscope (Zeiss, Berlin, Germany).The images were stored and submitted to a count ofinflammatory cells, evaluation of fibroblast prolifera-tion, analysis of the local angiogenesis and of the dia-meter of the ulcerated areas of the wounds at the endof the experiment, using digital marking.

Statistical analysisThe statistical analysis of the several parame-

ters evaluated in the two groups was made by a non-parametric method, the Mann-Whitney test, with a sig-nificance level of p<0.05.

RESULTSThe clinical observation of skin lesion samples

of the animals showed a small amount of clot on thesurface, with clear presence of blood vessels in thedeep hypodermis. These vessels, when accidentallyruptured in the dermis or hypodermis region, filledthe lesions partially or totally with clots.

On the 10th postoperative day, the skin lesionsof group I (control) exhibited an early-phase tissuerepair pattern, with formation of a whitish crust, withslightly elevated rims and a reddish core due to theaccentuated presence of blood irrigation in that area,indicating the presence of granulation tissue. On theother hand, the wounds of group II, which had beensubmitted to low-intensity laser treatment, showedcomplete tissue repair, exhibiting scars with evidentrims and a central portion slightly unleveled, but pre-senting an advanced morphological and functionalrecovery of the involved tissues.

The animals, placed in individual cages undermaintenance conditions which are in agreement withthe criteria of the Colégio Brasileiro deExperimentação Animal, were submitted to dailymacroscopic evaluations and presented no signs indi-cating secondary infection during the experiment.

In the analysis of the histological results, twodistinct areas were evaluated, one more superficial,involving epithelial proliferation, and the area of con-nective tissue beneath the more superficial portion ofthe lesion. In the control group, next to the rim of thesurgical wound, a discrete epithelial proliferation wasobserved, and virtually all over its extension the pre-sence of tissue exhibiting a broad ulceration area andfibrinonecrotic material over granulation tissue wasseen (Figure 1). However, in group II, the one submit-ted to laser therapy, the histopathological study evi-denced material showing undamaged epidermis cove-ring well-developed granulation tissue (Figure 2), withconnective tissue rich in collagen fibers with a parallelorientation with regard to the surface of the wound,determining a more organized tissue repair process.

Graph 1 presents the diameter of the ulceratedareas on the 10th postoperative day, the treated ani-mals having shown a significant reduction in the dia-meters of the ulcerated areas at the end of the expe-riment, as compared to the control group (p<0.05).As for vascularization, the treated animals presented asignificantly greater number of blood vessels permicroscopic field (Graph 2).

The histomorphometric analysis also demons-trated that the animals treated with LILT presented agreater number of fibroblasts per microscopic field(Graph 3), where the identification of fibroblasts,based on morphological criteria, was made by twodifferent researchers. Moreover, it was observed thatLILT significantly reduced the intensity of the inflam-matory infiltrate present in the lesions submitted totreatment (Graph 4).

DISCUSSIONLow-intensity laser treatment is a method that

is accepted by the Food and Drug Administration

152 Rocha Júnior AM, Oliveira RG, Farias RE, Andrade LCR, Aarestrup FM.


tions faced in the investigation of such contradictoryresults. LILT is still a controversial method, as can beseen in numerous publications,8,24,25 mainly becauseits mechanism of action is unknown.

The field of action of laser is very broad and thestudies showed its contribution to the tissue repairprocess, particularly regarding its influence on themodulation of certain cell types in the wound healingprocess. Gómez-Villamandos et al. reported an increa-se in wound healing after laser therapy, with increa-sed mitotic activity, number of fibroblasts, collagensynthesis and neovascularization of the injured tis-sues.26 Other authors16,27 observed that the production

of fibroblast growth factors (FGF) and the predomi-nance of fibroblasts in the culture increased conside-rably after low-intensity laser irradiation.Furthermore, Bisht et al. reported the developmentof granulation tissue and epithelization of wounds inWistar rats treated with He-Ne laser.28

In the present study, we evaluated vascular pro-liferation and observed a smaller amount of bloodvessels in the surgical lesions of the control group(Figure 3) compared to those of the treated group(Figure 4), where an increase in vascular proliferationwas found in the surgical wound samples, which wasalso observed when fibroblast proliferation in both


GRAPH 3: Total number of fibroblasts(10th postoperative day)

Results expressed in means ± standard deviation - p<0.05

GRAPH 4: Number of inflammatory cells (10th postoperative day)

FIGURE 3: Connective tissue showing few blood vessels in animalof the control group (blue arrows). HE staining, total

magnification 400 x

FIGURE 4: Connective tissue showing many blood vessels in ani-mal of the treated group (blue arrows). HE staining, total

magnification 400 x


groups was evaluated. In the control group samples,a smaller number of fibroblasts were observed(Figure 5) compared to those from the treated group,where a significant increase in fibroblast proliferationwas found (Figure 6).

Moreover, when the amount of inflammatorycells present in the lesions of both groups was analy-zed, a greater number of such cells were observed inthe control group samples (Figure 5) compared tothose from the treated group (Figure 6), which exhi-bited a significant decrease of inflammatory infiltrate.

These findings suggest a faster tissue repair inthe group submitted to LILT, expressed by an increa-

se in cell proliferation and vascularization, besides anexpressive decrease in the number of inflammatorycells.

CONCLUSIONTaken together, the results suggest that LILT is

effective in tissue wound healing, as observed in anexperimental model of surgical wounds produced inrats. This type of laser therapy showed positiveeffects, speeding up tissue proliferation, increasinglocal vascularization and forming a more organizedgranulation tissue. �

Modulation of fibroblast proliferation and inflammatory response... 155

FIGURE 5: Connective tissue showing a smaller number of fibrob-lasts (green arrows) and a large amount of mononucleated

inflammatory cells in the surgical wound of an animal of the con-trol group (blue arrows). HE staining, total magnification 400 x

FIGURE 6: Connective tissue showing a large number of fibroblasts(green arrows) and few mononucleated inflammatory cells in thesurgical wound of animal of the treated group (blue arrows). HE

staining, total magnification 400 x

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18.Gogia PP, Hurt BS, Zirn TT. Wound management with whirlpool and infrared cold laser treatment: a clinical report. Phys Ther. 1988;68:1239-42.

19.Lundeberg T, Malm M. Low-power He-Ne laser treatment of venous leg ulcers. Ann Plast Surg. 1991;27:537-9.

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MAILING ADDRESS:Prof. Fernando Monteiro AarestrupCentro de Biologia da Reprodução – CBRUniversidade Federal de Juiz de Fora - CampusUniversitário - Bairro Martelos 36036-330 - Juiz de Fora - MGTel.: +55 32 3229-3250 / Fax: +55 32 3229-3251E-mail: [email protected]



modulation of fibroblast proliferation and inflammatory response

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